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ABSTRACT: Advanced stages of tumour and development of metastases are the two major problems in treating liver tumours such as hepatoblastoma (HB) and hepatocellular carcinoma (HCC), in paediatric patients. Modulation of apoptosis in HB cells enhances the sensitivity of these cells towards various drugs and has been discussed to enforce treatment. We analysed the effect of apoptosis modulators, BH3 mimetics, on mechanisms of dissemination such as adhesion or migration of HB and HCC cells. BH3 mimetics such as ABT-737 and obatoclax can reduce cell migration in a scratch assay as well as adhesion of HB and HCC cells to matrigel. Immunofluorescence staining of F-actin demonstrated that development of lamellipodia, which are important for migration, decreased. BH3 mimetics increase the level of activated caspases 3 and 7 in HUH6 cells. This results in the degradation of GTPase Cdc42, which can be determined by western blot analysis. A pan-caspase inhibitor can block the migration and degradation of Rho-GTPase. In summary, our study showed that BH3 mimetics not only enhance drug sensitivity but also may prevent metastasis by inhibiting HB and HCC cell motility.
Experimental Cell Research 02/2013; · 3.58 Impact Factor
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ABSTRACT: Hepatoblastoma (HB) is the most common liver cancer in children. Recurrence of HB after chemotherapy and surgery is frequent among high-risk patients and is associated with chemoresistance. Immunotherapy may improve poor treatment outcomes in HB patients. Cytotoxic leukocytes of the innate and adaptive immune system including different populations of cytotoxic T cells play a major role in fighting developing tumors. In this setting, monoclonal antibodies may be employed to specifically direct immune responses toward tumor cells. We addressed this issue by using humanized antibodies that recognize the cell surface molecule EpCAM (CD326, overexpressed in hepatic tumor cells) to enhance immune responses against HB. EpCAM was constantly expressed on HB cells and its expression was independent of previous therapy based on the DNA-damaging agent cisplatin. Co-culture assays performed with two well-described HB cell lines and tumor tissue cultures demonstrated that tumor cell lysis by γδ T cells can be dramatically augmented by applying EpCAM-specific monoclonal antibodies. These data emphasize the value of antitumor immune responses and encourage adapting immunotherapeutic regimens to improve the outcome of high risk HB.
Oncoimmunology. 01/2013; 2(1):e22620.
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ABSTRACT: PURPOSE: Duplex drugs are promising anticancer agents. After in vivo cleavage into active nucleoside analogues, they exert their anti-tumour activity with reduced toxicity and side effects. Here we evaluated the impact of two duplex drugs on the viability of hepatoblastoma (HB) cells lines and their toxicity against human fibroblasts. METHODS: The duplex drugs 2'-deoxy-5-fluorouridylyl-(3'-5')- 3'-C-ethynylcytidine (5-FdU(3'-5')ECyd) and 3'-C-ethynylcytidinylyl-(5'→1-O)-2-O-octadecyl-sn-glycerylyl-(3'-Ο→5')-2'-deoxy-5-fluorouridine (ECyd-lipid-5-FdU) were analysed in two HB cell lines (HUH6, HepT1) and fibroblasts by MTT assay. The treatment potential was compared to the single substances 2'-deoxy-5-fluorourindine (5-FdU), 3'-C-ethynylycytidine (ECyd) and an equimolar mixture of both. Cell cycle analyses were performed using flow cytometry after 7-AAD staining. RESULTS: Both duplex drugs achieve a potent cytotoxic effect at low μM concentrations, which was more pronounced than the mixture of ECyd + 5-FdU. Further, both substances exert toxicity on fibroblasts of tumour samples, with less toxicity in foreskin fibroblasts cultures. Cell cycle analyses revealed a shift towards apoptotic cells for both drugs in HB cells. CONCLUSION: 5-FdU(3'-5')ECyd and ECyd-lipid-5-FdU exert a highly potent anti-tumoural effect on HB cells and might therefore be a treatment option in HB. Pharmacological formulations of both duplex drugs have to be evaluated in vivo to reduce possible side effects.
Pediatric Surgery International 11/2012; · 1.25 Impact Factor
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ABSTRACT: The response of standard-risk hepatoblastoma (HB) to neoadjuvant cisplatin (CDDP) chemotherapy is excellent; however, in high-risk HB, drug resistance remains a major challenge. Alternative therapeutic strategies may consider combining cytotoxic drugs with apoptosis sensitizers as this has shown additive effects in various types of malignancies. Analysis of published expression databases have revealed an anti-apoptosis state in HB samples. Herein, we evaluated the synergistic effects of ABT-737 as a modulator of apoptosis in combination with CDDP in HB. To this end, clonogenic assays were performed with HepT1 and HUH6 HB cells to evaluate the synergistic effects of CDDP and ABT-737. Combination treatment with CDDP and ABT-737 reduced the clonogenicity of HB cells more than 5-fold compared to treatment with CDDP alone. Furthermore, the HUH6 mixed-type HB cells showed higher sensitivity to CDDP and combination treatment compared to the HepT1 embryonal-type cells. Subcutaneous HUH6 tumors in NOD/LtSz-scid IL2Rγnull mice were treated with CDDP (1.25 and 3 mg/kg body weight, n=6), ABT-737 (100 mg/kg, n=5) and the combination of both agents (n=5). Combined treatment led to a significantly reduced tumor growth compared to CDDP treatment alone (p<0.02). When using higher doses of CDDP (3 mg/kg) alone or in combination with ABT-737, dose-dependent toxicity was observed in this mouse strain. In conclusion, our results demonstrated the enhancement of chemotherapy efficacy by using modulators of apoptosis together with cytotoxic agents. Additive effects of ABT-737 may allow reduction in CDDP dosages with maintenance of antitumor activity. Sensitizing HB to apoptosis may also render resistant HB susceptible to established chemotherapy regimens.
Oncology Reports 11/2012; · 1.84 Impact Factor
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ABSTRACT: Animal models, including xenografts, models of metastatic invasion, syngeneic models and transgenic models, are important tools for basic research in solid pediatric tumors, while humanized animal models are useful for novel immunotherapeutical approaches. Optical and molecular imaging techniques are used for in vivo imaging and may be used in conjunction with alternative treatment approaches, including photodynamic therapy. The aim of this review is to highlight the various animal models that may be used for basic research in pediatric solid tumors.
Oncology letters 11/2012; 4(5):859-864. · 0.11 Impact Factor
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ABSTRACT: BACKGROUND: Chemoresistance and advanced tumour stage at time of diagnosis are the major reasons for poor treatment results in hepatoblastoma (HB) and paediatric hepatocellular carcinoma (HCC). Positive results with transplantation of liver and bone marrow revealed the impact of the immune system on the treatment of liver malignancies. AIM: Cytotoxic-immune-cells-like natural killer (NK) and T cells are major player in the defence against developing tumours. This study aimed to specifically analyse the ability of ex-vivo expanded γδ T cells to recognise and lyse HB and HCC cell lines in coculture assays. METHODS: Cell viability after treatment with γδ T cells was evaluated with two HB (HUH6 and HepT1) and one HCC cell line (HC-AFW1) using a MTT-based cytotoxicity assay. The binding of T cells to target cells was monitored using immunofluorescence microscopy. RESULTS: Incubation of hepatic tumour cell lines with γδ T cells led to a significant decrease in tumour cell viability. This was enhanced by zoledronic acid and histone deacetylase inhibitors. MT110, an EpCAM/CD3-bispecific BiTE antibody could bluntly enhance tumour cell lysis close to completion. γδ T cells efficiently interacted with HB and HCC cells in a spheroid culture model. CONCLUSION: Bispecific antibodies such as MT110 might be used to intensify the antitumoural effect of γδ T cells in context of adoptive immune cell transfer. Optimised immunotherapeutic strategies might therefore improve the outcome of high risk hepatoblastoma and hepatocellular carcinoma.
Liver international: official journal of the International Association for the Study of the Liver 10/2012; · 3.82 Impact Factor
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ABSTRACT: The prognosis of advanced stage rhabdomyosarcoma (RMS) is still sobering. In recent years, outcome has not been further improved by conventional therapy. Therefore, novel treatment options such as macrophage-directed immunotherapy have to be investigated. The aim of this study was to analyze the phagocytosis of RMS cells by macrophages and to modulate the susceptibility using monoclonal antibodies and cytotoxic drugs. Expression of the macrophage activating ligand calreticulin and CD47, the counterpart of the inhibitory receptor SIRPα, was analyzed with Affymetrix mRNA expression arrays and immunohistochemistry on 11 primary RMS samples. Results were verified in two RMS cell lines using flow cytometry and immunocytochemistry. Macrophage cytotoxic activity was quantified by a MTT colorimetric assay in co-culture experiments of RMS cells with monocyte-derived, GM-CSF stimulated macrophages. Gene expression analysis and immunohistochemistry revealed a high expression of CD47 and calreticulin in alveolar and embryonal RMS tissue specimens. Extracellular expression of CD47 on RMS cell lines was confirmed by flow cytometry, whereas calreticulin was exclusively detected in the endoplasmatic reticulum. After co-culturing of RMS cells with macrophages, viability dropped to 50-60%. Macrophage-mediated cytotoxicity was not influenced by a blocking antibody against CD47. However, susceptibility was significantly enhanced after pre-treatment of RMS cells with the anthracycline drug doxorubicin. Furthermore, translocation of calreticulin onto the cell surface was detected by flow cytometry. The immunologic effect of doxorubicin may improve the efficacy of adoptive cellular immunotherapy and chemotherapy of childhood RMS.
Oncoimmunology. 05/2012; 1(3):279-286.
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ABSTRACT: Hepatoblastoma (HB) has a poor prognosis in advanced stages. The aim of this study was to enhance effectiveness of chemotherapy with antineoplastic kinase inhibitors.
Viability was monitored in HB cells (HUH6, HepT1) in monolayer and spheroid cultures treated with kinase inhibitors VX-680, Wee1-InhibitorII, and SU11274 alone or in combination with cisplatin (CDDP) using MTT assays. Apoptosis was revealed by Caspase-3 assay. Western blot and immunohistochemical analyses were performed to determine histone H3 phosphorylation.
Among the kinase inhibitors strongest anti-proliferative effect on HB cells was documented for VX-680. HUH6 cells responded more sensitively to the Aurora kinase inhibitor as HepT1 cells (IC(50) 8 and 16.6 μM, respectively). While VX-680 and CDDP showed no additive effects, the combination of VX-680 and histone deacetylase inhibitor SAHA had a synergistic effect on the proliferation of HUH6 cells. The inhibition with VX-680 led to reduced histone H3 phosphorylation, to an increase of apoptotic cells, and to morphological changes such as vacuolization and swelling of the cells and nuclei.
The data provide evidence that VX-680 might improve treatment results in HB with increased Aurora kinase activity by inhibiting cell proliferation and induction of apoptosis.
Pediatric Surgery International 04/2012; 28(6):579-89. · 1.25 Impact Factor
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ABSTRACT: Drug resistance remains a major challenge for the treatment of high-risk hepatoblastoma (HB). To enhance effectiveness of chemotherapy we modulate apoptosis in HB cells in vitro.
Viability was monitored in HB cells (HuH6, HepT1) and fibroblasts in monolayer and spheroid cultures treated with ABT-737, obatoclax, HA14-1, and TW-37 and each in combination with CDDP, etoposide, irinotecan, paclitaxel, and DOXO in a MTT assay. Western blot analyses were performed to determine expressions of pro- and anti-apoptotic proteins.
Obatoclax and ABT-737 led to a dose-dependent decrease of viability in HB cells at concentrations above 0.3 μM. TW-37 and HA14-1 were less effective. ABT-737 and obatoclax had additive effects when combined with CDDP, etoposide, irinotecan, paclitaxel, or DOXO. This was also observed for fibroblast, however, for higher drug concentrations. In spheroid cultures, relative expression of Bcl-XL was increased, Bax was decreased, Mcl-1 was low, and Bcl-2 was not detected compared to 2D cultures, denoting an anti-apoptotic state in spheroids. Obatoclax and ABT-737 have overcome the resistance to CDDP. HuH6 cells have shown higher susceptability for apoptosis sensitizers than HepT1.
The data provide evidence that ABT-737 and obatoclax might improve treatment results in children with HB.
Pediatric Surgery International 02/2012; 28(2):149-59. · 1.25 Impact Factor
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ABSTRACT: Current treatment of paediatric hepatocellular carcinoma (HCC) is often inefficient due to advanced disease at diagnosis and resistance to common drugs. The aim of this study was to generate a cell line derived from a paediatric HCC in order to expand research in this field. We established the HC-AFW1 cell line from a liver neoplasm of a 4-year-old boy through culturing of primary tumor specimens. The cell line has been stable for over one year of culturing and has a doubling time of 40 h. The tumour cells have an epithelial histology and express HCC-associated proteins such as Alpha-fetoprotein (AFP), Glypican 3, E-cadherin, CD10, CD326, HepPar1 and Vimentin. Forty-nine amino acids in exon 3 of β-Catenin that involve the phosphorylation sites of GSK3 were absent and β-Catenin is detectable in the cell nuclei. Cytogenetic analysis revealed large anomalies in the chromosomal map. Several alterations of gene copy numbers were detected by genome-wide SNP array. Among the different drugs tested, cisplatin and irinotecan showed effective inhibition of tumour cell growth in a proliferation assay at concentrations below 5 µg/ml. Subcutaneous xenotransplantation of HC-AFW1 cells into NOD/SCID mice resulted in fast growing dedifferentiated tumours with high levels of serum AFP. Histological analyses of the primary tumour and xenografts included national and international expert pathological review. Consensus reading characterised the primary tumour and the HC-AFW1-derived tumours as HCC. HC-AFW1 is the first cell line derived from a paediatric HCC without a background of viral hepatitis or cirrhosis and represents a valuable tool for investigating the biology of and therapeutic strategies for childhood HCC.
PLoS ONE 01/2012; 7(5):e38223. · 4.09 Impact Factor
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ABSTRACT: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is responsible for cell death in many cancer cells while being non-toxic for most normal cells. In this study, we investigated the role of TRAIL in human hepatoblastoma (HB) cells and analyzed different approaches to reverse TRAIL resistance in these tumors. Death receptors DR4 and DR5 expression was found on all analyzed primary HB samples and on the cell lines HuH6 and HepT1 by immunofluorescence staining. Recombinant TRAIL alone did not induce in vitro cytotoxicity. Decoy receptor blocking by antibodies led to moderate effects in HepT1 but not in HUH6 cells, whereas FLIP knock-down using siRNA rendered HUH6 cells but not HepT1 cells sensible to TRAIL. Bcl-2 inhibition with ABT-737 enhanced TRAIL-mediated apoptosis in all HB cells. Strongest cytotoxic TRAIL effects were seen in HB cell lines with synchronous proteasome inhibition using bortezomib. FLIP and Bcl-2 contributed to the TRAIL resistance in HB. Overcoming TRAIL resistance in HB by proteasome inhibitors has been identified a possible additive to improve treatment results in HB patients with drug resistant tumors.
Frontiers in bioscience (Elite edition) 01/2012; 4:2194-202.
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ABSTRACT: Multidrug resistance is a major reason for poor treatment results in advanced hepatoblastoma (HB). Several alternative treatment options are currently under investigation to improve the prognosis of affected patients
This study aimed to analyse the impact of sorafenib on the viability of HB cells and xenotransplanted HB tumours.
Cell viability and apoptosis were evaluated in two HB cell lines (HUH6 and HepT1) after treatment with sorafenib using MTT and Caspase 3 activation assay. Extracellular signal-regulated kinase (ERK) phosphorylation was investigated using Western blot. In addition, sorafenib (30 mg/kg) was administered orally to NMRI mice bearing subcutaneous HUH6 derived tumours. Tumour progression and viability were monitored by tumour volume and α-fetoprotein (AFP) levels, and apoptosis was assessed using TUNEL assay. Tumour angiogenesis and mean vascular density (MVD) was determined using CD31 staining, ERK phosphorylation was detected using indirect immunofluorescence.
Treatment with sorafenib led to decreased ERK phosphorylation, reduced cell viability and induction of apoptosis in HepT1 and HUH6 cells. In HB xenografts, sorafenib significantly reduced tumour growth compared with control (P < 0.05). AFP levels were lower in the sorafenib group (P = 0.07). Relative apoptotic areas detected using TUNEL assay were increased (P = 0.003). CD31 staining revealed inhibition of angiogenesis, and mean vascular density was lower in the sorafenib group (P = 0.02). ERK phosphorylation was reduced in tumours tissues after sorafenib treatment.
Treatment with sorafenib led to a potent inhibition of cell viability, tumour progression and angiogenesis. Sorafenib might therefore also be a promising treatment option for high risk or recurrent HB.
Liver international: official journal of the International Association for the Study of the Liver 12/2011; 32(4):574-81. · 3.82 Impact Factor
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ABSTRACT: The primary goal of current chemotherapy in hepatoblastoma (HB) is reduction of tumour volume and vitality to enable complete surgical resection and reduce risk of recurrence or metastatic disease. Drug resistance remains a major challenge for HB treatment. In some malignancies inhibition of anti-apoptotic pathways using small BH3 mimetic molecules like ABT-737 shows synergistic effects in combination with cystotoxic agents in vitro. Now we analysed toxicology and synergistic effects of this approach in HB cells and HB xenografts.
Viability was monitored in HB cells (HUH6 and HepT1) and fibroblasts treated with paclitaxel, ABT-737 and a combination of both in a MTT assay. HUH6 xenotransplants in NOD/LtSz-scid IL2Rγnull mice (NSG) were treated accordingly. Tumour volume and body weight were monitored. Xenografted tumours were analysed by histology and immunohistochemistry (Ki-67 and TUNEL assay).
ABT-737 reduced viability in HUH6 and HepT1 cells cultures at concentrations above 1 μM and also enhanced the cytotoxic effect of paclitaxel when used in combination. Thereby paclitaxel could be reduced tenfold to achieve similar reduction of viability of tumour cells. In contrast no toxicity in fibroblasts was observed at the same regiments. Subcutaneous HB (HUH6) treated with paclitaxel (12 mg/kg body weight, n = 7) led to delayed tumour growth in the beginning of the experiment. However, tumour volume was similar to controls (n = 5) at day 25. Combination treatment with paclitaxel and ABT-737 (100 mg/kg, n = 8) revealed significantly 10 fold lower relative tumour volumes compared to control and paclitaxel groups. Paclitaxel dependent toxicity was observed in this mice strain.
Our results demonstrate enhancement of chemotherapy by using modulators of apoptosis. Further analyses should include improved pharmacological formulations of paclitaxel and BH3 mimetics in order to reduce toxicological effects. Sensitising HB to apoptosis may also render resistant HB susceptible to established chemotherapy regimens.
BMC Cancer 08/2011; 11:362. · 3.01 Impact Factor
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ABSTRACT: Expression profiling of tumor tissue allows a systematic search for targeted therapies and offers relevant prognostic information. Molecular studies on rhabdomyosarcoma (RMS) revealed a more differentiated classification than the histological subgrouping into embryonal (RME) and alveolar (RMA) rhabdomyosarcoma, and reflected the chromosomal aberrations found in RMS. We addressed biological processes like cell migration and emerging drug resistance by expression profiling to identify mechanisms of metastasic invasion and differential response to chemotherapy in RMS. Gene expression analysis was performed in 19 RMS samples using the Affymetrix U133 Plus2 array. Validation of target genes was performed by qRT-PCR. Data were analyzed using Pathway analysis software. Involvement of these genes in invasion processes was evaluated in knock-down experiments using specific interference RNA and Matrigel(TM) invasion assay. In RMA tissues 211 of 534 genes were overexpressed, in RME tissues 323 genes were overexpressed. Pathway analysis software identified a group of genes involved in cell growth, morphology and motility. In patients with distant metastases especially transcription factors such as FOXF1 and LMO4 showed a high expression, which were described as determinants of tumor cell migration. Down-regulation of these factors inhibited the invasion of RMS cells >10-fold. Microarray technology is a powerful method not only to classify RMS samples, but also to identify major regulatory processes. Functional evaluation of LMO4 and FOXF1 identified targets of a molecular network for preventing metastatic invasion in RMS.
International Journal of Oncology 04/2011; 38(4):993-1000. · 2.40 Impact Factor
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ABSTRACT: Carcinogenesis is often linked to aberrant activation of Wnt/β-catenin signalling, in many cases caused by activating CTNNB1 mutations (encoding β-catenin). Recently, β-catenin was established as a decisive regulator of hepatic glutamine synthetase (GS) and cytochrome P450 (CYP) expression in mouse hepatocarcinogenesis. This study was aimed to analyse the connection of β-catenin signalling and GS/CYP expression in human paediatric tumours. Samples from 23 paediatric tumours were analysed for activating mutations in CTNNB1. Protein expression of the model β-catenin target GS and of various CYP isoforms was analysed and correlated with CTNNB1 mutational status and histological findings. Activating CTNNB1 mutations were frequent in hepatoblastoma (80%) and nephroblastoma (31%). In CTNNB1-mutated hepatoblastoma, expression of GS was only detected in tumour areas with epithelial, not with mesenchymal differentiation. Particularly high expression of glutamine synthetase was found in hepatoblastoma cells directly neighbouring a mesenchymal-type tumour area or stroma cells, associated with above-average cell proliferation. GS expression was not observed in CTNNB1-mutated nephroblastoma. Hepatoblastoma with activated β-catenin expressed different CYPs relevant for the metabolism of cytostatic drugs, but with high interindividual variance and heterogeneity within a single tumour. GS and different CYPs are co-expressed in hepatoblastoma with activated β-catenin. Moreover, other factors like histological subtype of tumour cells and cell-cell-interactions at the borders between different areas of the tumours affect expression of these β-catenin target genes. Analysis of CYP expression in resected tumour tissue might be useful for the selection of appropriate cytostatics for post-operative chemotherapy.
Toxicology 03/2011; 281(1-3):7-14. · 3.68 Impact Factor
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ABSTRACT: Multidrug resistance (MDR) is a major reason for poor treatment results in hepatoblastoma (HB). The objective of this study was to establish a drug resistance model for HB to analyse alternative treatment options in vitro. Both HB cell lines HUH6 and HepT1 were xenotransplanted in NMRI mice (nu/nu) and 2 cycles of cisplatin (CDDP) treatment were administered. Thereafter, xenotransplants were excised and viable tumour cells were re-cultured. 3D cultures of HUH6 and HepT1 cells were generated on a low binding culture surface. Cell viability in response to CDDP/DOXO (doxorubicin) and apoptosis was assessed by MTT-assay and caspase 3 activity, respectively. Efflux of doxorubicin was measured by flow cytometry. Cellular levels of ABC-transporters (MDR1, MRP1, cMOAT and BRCP) were determined by real time rt-PCR. Only HepT1 cells isolated from HB xenografts showed resistance to CDDP, but did not survive repeated passages. Culturing HUH6 and HepT1 cells as spheroids was successful and 3D cultures showed an IC50-drift to higher drug concentrations for CDDP and DOXO compared to 2D cultures. Treatment with CDDP and DOXO led to homogeneous apoptosis in spheroids. Increased doxorubicin efflux in HUH6 spheroids was not influenced by the P-glycoprotein inhibitor tariquidar. Expression levels of MDR1, MRP1, cMOAT and BRCP in 3D cultures were similar to those in 2D cultures and were higher in HepT1 than in HUH6 cells. In conclusion, a 3D cell culture model for multidrug resistance was established for hepatoblastoma. The underlying mechanism involves altered accessibility of the cells for drugs rather than up-regulation of ABC-transporters.
International Journal of Oncology 02/2011; 38(2):447-54. · 2.40 Impact Factor
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ABSTRACT: Investigation of hepatoblastoma in experimental conditions contributes relevantly to a detailed understanding of tumor biology and the investigation of new treatment approaches. Most systematical analyses currently use subcutaneous xenografts. We established a reproducible intrahepatic model with the hepatoblastoma-cell lines HuH6 and HepT1. The cells were stably transfected with a plasmid vector encoding for Gaussia luciferase. HuH6 and HepT1 were injected intrasplenically in NOD/LtSz-scid IL2Rγnull mice. Mice were splenectomized in order to avoid intrasplenical tumor growth. Multifocal intrahepatic tumor growth was observed in 85% (11/13) of HuH6 tumors and 55% (5/9) of HepT1 tumors. Serum Alpha-fetoprotein and Gaussia luciferase increased 5 weeks after tumor-cell inoculation. Tumors were detected by MRI at this time point. Immunhistochemical analysis such as vascularity (CD31), proliferation index (Ki-67), cytokeratin 7 and distribution of β-catenin in intrahepatic tumors were different to subcutaneous tumors. We established a reproducible xenograft model for intrahepatic hepatoblastoma growth with a high tumor incidence. Monitoring of tumor cell viability was optimized by measuring GLuc. This model enables further experimental investigations of HB in a more physiological milieu as emphasized by the β-catenin distribution.
PLoS ONE 01/2011; 6(8):e23419. · 4.09 Impact Factor
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ABSTRACT: An increased expression of anti-apoptotic proteins is regularly found in malignant cells, contributing to their clonal expansion by conferring an improved survival ability. In Hepatoblastoma (HB) apoptosis regulation contributes to resistance and therapy failure, therefore we modulated apoptosis sensitivity of HB cells for an improved cytotoxic activity of commonly used drugs.
Apoptosis-related proteins were quantified in HB cells (HuH6 and HepT1) using protein assays. Interaction of ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xL, and Bcl-W with cytotoxic drugs was monitored in a proliferation assay. Apoptosis induction was measured by caspase-3 activity.
We found high levels of the anti-apoptotic protein Bcl-2 and Bcl-X as well as low levels of pro-apoptotic protein Bax and Bad in both HB cell lines. ABT-737 induced apoptosis in HuH6 and HepT1 cells at concentrations higher than 1 µM. ABT-737 also enhanced the cytotoxic effect of cisplatin (CDDP), doxorubicin (DOXO), etoposide and paclitaxel when used as combination therapy. HuH6 expressed slightly higher pro-apoptotic and lower anti-apoptotic protein levels than HepT1, which may explain the stronger enhancement of cytostatic drug effects in HuH6 cells when treated in combination with ABT-737.
The observed anti-apoptotic phenotype in HB cell lines may contribute to resistance to cytotoxic drugs used in the standard treatment protocol of HB. These pre-clinical results suggest that apoptosis sensitizers with BH-3 mimicry, such as ABT-737, should be further evaluated in preclinical models of HB.
Pediatric Blood & Cancer 12/2010; 55(6):1089-95. · 1.89 Impact Factor
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ABSTRACT: The outcome of patients with advanced stage rhabdomyosarcoma (RMS) is still sobering. This outcome has not improved through conservative treatments. Therefore, novel treatment approaches such as immunotherapy need to be evaluated in human-adapted animal models. The aim of this study was to develop a humanized mouse model of childhood RMS as a basis for the study of immunotherapeutic approaches. Therefore, NOD/LtSz-scid IL2rgammanull-mice were used for all the experiments (n=19). The animals underwent sublethal irradiation on days 1 and 2 (1 x 300 cGy). After irradiation, the transplantation of human CD34+-cells (1,000,000 cells per animal i.v.) was carried out. Five animals served as the control and did not undergo stem cell transplantation. The engraftment of human cells was assessed in peripheral blood on days 21 and 55 by FACS analysis. Eight weeks after transplantation, the subcutaneous xenotransplantation of human alveolar and embryonal RMS cell lines was carried out. Tumor growth was monitored and tumors were resected 93 days after CD34+-transplantation. The tumor specimens were evaluated histologically. The successful engraftment of human cells with the establishment of a human immune system was observed in 12 out of 14 animals. B and T cells were mostly detected in the peripheral blood. There were only a few monocytes and almost no natural killer cells. The xenotransplantation of alveolar RMS resulting in subcutaneous tumor growth was feasible in 7 animals. The xenotransplantation of embryonal RMS was performed in 5 animals and led to tumor growth in 1 animal. A histological work up showed either alveolar or embryonal RMS cells with central necrosis. This is the first time a xenotransplantation model of human RMS has been developed in a humanized mouse model. The establishment of subcutaneous tumor xenografts was more effective in the alveolar subtype. This model offers a basic tool for further analyzing novel immunotherapeutic approaches in RMS, and could possibly be used in other solid pediatric tumors.
Oncology Reports 10/2010; 24(4):1067-72. · 1.84 Impact Factor
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ABSTRACT: Multidrug resistance is a common problem in the treatment of childhood rhabdomyosarcoma (RMS). Therefore, novel treatment regimes such as immunotherapy have to be evaluated. The aim of this study was to detect possible targets on RMS cells and to investigate whether corresponding humanized antibodies could be used to treat RMS. Screening for potential targets common for different subtypes of RMS was carried out with Affymetrix mRNA expression arrays on 12 primary RMS samples. Subsequent pathway analysis revealed the epidermal growth factor receptor (EGFR) as a potential target for antibody therapy. Expression of EGFR and binding of its specific antibody Cetuximab to embryonal RMS cell lines RD and A-204 and alveolar RMS Rh30 were monitored by flow cytometry. Cetuximab activity was quantified by proliferation assay on RMS cells, and by antibody dependent cellular cytotoxicity assay with peripheral blood mononuclear cells (PBMCs). Gene expression analysis revealed a high expression of EGFR in all embryonal RMS compared with alveolar RMS. The EGFR specific antibody, Cetuximab binds to Rh30 and to RD but not to A-204 cells. Proliferation of these cells was influenced neither by Cetuximab nor by the growth factor EGF. However, cell dependent cytotoxicity of PBMCs to RMS cells such as Rh30 and RD was enhanced specifically by Cetuximab. These promising Cetuximab effects justify analysis under in vivo conditions using suitable models.
Journal of immunotherapy (Hagerstown, Md.: 1997) 04/2010; 33(3):279-86. · 3.20 Impact Factor
